Spontaneous ribosome bypassing in growing cells

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Translating ribosomes can skip sections of messenger RNA (mRNA) and resume translation later.
• Previous studies focused on bypass during amino acid starvation.
• The frequency and conditions of spontaneous mRNA bypassing in unstarved cells were less understood.

Purpose of the Study:

• To investigate spontaneous messenger RNA (mRNA) bypassing in unstarved, growing cells.
• To determine the factors influencing the frequency of ribosome takeoff and bypassing.
• To estimate the overall efficiency of translation termination and bypassing.

Main Methods:

• Constructed lacZ reporter systems with various sense codons as takeoff sites and landing sites 16 nucleotides downstream.
• Measured beta-galactosidase synthesis to quantify bypassing frequency.
• Used sequencing (Edman degradation, mass spectrometry) to confirm bypass events.
• Varied tRNA abundance by introducing additional tRNA genes to assess its effect on bypassing.

Main Results:

• Spontaneous bypassing occurs in unstarved cells, with significant variation based on takeoff codons (e.g., UUU, AGG).
• Bypassing frequency inversely correlated with the abundance of tRNA corresponding to the A-site codon.
• Increased tRNA gene copy number reduced spontaneous bypassing.
• Estimated the average ribosome takeoff frequency and calculated a translation success probability of approximately 0.73.

Conclusions:

• Ribosome bypassing is a spontaneous event in growing cells, not solely a response to starvation.
• Empty ribosome A-sites, caused by low tRNA levels or pauses, stimulate bypassing.
• These findings provide insights into translation efficiency and potential regulatory mechanisms.